Advances in high-throughput sequencing (HTS) have accelerated the discovery of the genetic basis of numerous human diseases and conditions, including congenital disorders. These conditions affect a wide range of physiological systems, including cardiovascular, craniofacial, nervous, genital/urinary, in addition to general defects growth and dysmorphology. However, the process of ascribing causality to a given variant can be challenging and remains a major bottleneck in our understanding of human disease. Animal model validation is a powerful tool to evaluate the causality of a given variant, and the mouse provides the ideal mammalian system to model developmental disorders. CRISPR/Cas9 technology has simplified and reduced the timelines for mouse model creation, but breeding time will still typically take up to a year or more before phenotypes can be examined. Thus, a validation platform that directly examines this founder population (F0 generation) promises to massively reduce the time and expense required for validation of new human disease mutations in the mouse. Our preliminary work provides proof-of-principle evidence that this approach is feasible, however challenges remain before such an approach can be applied to a wide range of human developmental conditions. Direct screening of F0 embryos provides a means to examine more difficult engineering and breeding challenges, include dominant mutations that are predicted to be lethal in the mouse, and modeling multigenic causes of disease. Moreover, a F0 platform can provide a tool for rapid, direct genetic interrogation of pathways through multiplex mutagenesis. Challenges remain, however, before this approach can be effectively implemented as a robust platform. For example, while generation indels and deletions through error-prone non-homologous end joining (NHEJ) repair is highly efficient, editing of specific mutations through homology directed repair (HDR) occurs at a much lower rate. Additionally, founders and F0 embryos are typically mosaic and the tools to quantitatively assess both the spectrum of alleles and the mutagenesis rate for each require further development. Therefore, the overarching goal of this proposal is to build upon our proof-of-principle studies to optimize our F0 screening platform and implement it to model structural birth defects. We will test our hypotheses that 1) modification of parameters of the CRISPR mutagenesis protocol can improve the efficiency and reduce the mosaicism of HDR; and 2) that we can apply the platform to specific unique congenital disease challenges not easily modeled with standard mouse genetics approaches. This will take advantage of several key resources, including a high-throughput embryonic phenotyping pipeline developed for the Knockout Mouse Phenotyping Program (KOMP2) and the large-scale mouse production capabilities of The Jackson Laboratory. The long-term goal is to integrate our screening platform with large- scale precision modeling initiatives, providing a rapid and robust means to evaluate new genetic variants in a mammalian model system.